The aim of the proposed research is to determine the mechanism by which energy transducing membrane proteins can store free energy in the form of a transmembrane electrochemical potential gradient for protons. The chief experimental system we will use is bacteriorhodopsin (bR), a light-driven proton pump. There is mounting evidence from both respiratory and photosynthetic systems that the translocated protons are at least partially confined to and conducted by charged buffering groups at the membrane surface. Our approach is to use modern biophysical methods to directly measure the protons and other ions which move during the cycle of operation of the proton pump. Specifically, we will employ a very sensitive differential ac conductivity bridge developed in our laboratory for the detection of transient ion movements. The apparatus can detect these small changes in ionic concentration in the presence of molar concentrations of electrolyte, buffered solution and for good measure it gives te absolute quantum yield. We plan to compare the results with isolated bR to those with bR in vesicles and whole cells to study the coupling of the protonmotive force to energy-consuming functions such as ATP synthesis and active transport. Understanding this process is critical to understanding how living systems maintain the energy flow required for cellular growth, organization and reproduction. We also plan collaborative experiments using our conductivity method to determine the kinetics, pH dependence and quantum yield of proton uptake and release from photo-excited bacterial reaction centers and the oxygen evolution system from algae.